Systems and methods for raman spectroscopy

ABSTRACT

A method of performing Raman spectroscopy can include guiding a Raman pump beam with an optical fiber, where the Raman pump beam inducing fluorescence in the optical fiber. The beam and the fluorescence are coupled to a photonic integrated circuit (PIC) via the fiber. The beam is used to excite a sample in optical communication with the PIC via evanescent coupling and induces Raman scattering in the sample. The Raman scattering is collected via the PIC, and the Raman pump beam as well as the fluorescence is filtered out from the Raman scattering via the PIC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/691,073 filed Jun. 28, 2018, titled “SYSTEMS AND METHODS FOR RAMANSPECTROSCOPY OF CHEMICAL AND BIOLOGICAL SPECIES USING PHOTONICINTEGRATED CIRCUITS”, the entire disclosure of which is incorporatedherein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.ECCS-1709212 awarded by the National Science Foundation. The Governmenthas certain rights in the invention.

BACKGROUND

A Raman spectrometer or Raman spectrophotometer is a device thatoptically probes the vibrational, rotational, and low-frequency modes ofa solid, liquid, or gaseous chemical or material. Raman spectroscopy isa technique for accurately quantifying the chemical structure of anunknown substance. Raman spectroscopy typically involves firstilluminating a sample with a single frequency laser in the visible ornear-infrared wavelength region. A fraction of the light scattered bythe sample is converted to a higher optical frequency (anti-Stokesshifted), and another fraction is converted down to a lower opticalfrequency (Stokes shifted), with these frequencies corresponding to theintrinsic energy levels of the substance being sensed, as shown inFIG. 1. By comparing the measured spectrum to a database of known Ramanspectra, the composition of single chemicals or mixtures of chemicals inthe gas, liquid, or solid phase can be determined with high precision.

A conventional Raman spectrometer system may comprise several distinctsub-systems, including: (1) a single-frequency excitation source, suchas a laser; (2) an optical filter that suppresses amplified spontaneousemission at wavelengths above and below the laser wavelength; (3) anoptical probe or region where the light interacts with the analyte orunknown chemical of interest; (4) a dichroic mirror or optical filterthat removes the light from the excitation source, letting the Stokes oranti-Stokes scattered light pass; and (5) a spectrum analyzer orspectrometer that measures the intensity of the Raman shifted light as afunction of frequency or wavelength.

The waveguide-enhanced Raman spectroscopy (WERS) technique has recentlybeen demonstrated for detecting chemical and biological species intightly confined single-mode waveguides on a chip. However, practicalimplementations of this technology require fiber-coupled light to bedelivered to the chip, but the presence of the silica fiber producessignificant background noise in the form of fluorescence and Ramanscattering from the fiber material.

Additionally, the pharmaceutical industry has traditionally relied onbatch manufacturing to produce molecules of interest. The high fixedcosts and low flexibility associated with the use of large, multi-usevessels have recently driven a shift towards new ways of chemicalproduction, such as continuous manufacturing, where the productionprocess is performed all at once along tubes and smaller vessels, andsingle-use technologies, which rely on disposable, smaller-volumeequipment. Yet conventional analytical tools, which typically allow forthe measurement of one sample at a time and whose cost prevents singleusage, are hardly adapted to this new manufacturing paradigm.

SUMMARY

In some aspects, a photonic integrated circuit (PIC) for Ramanspectroscopy includes a semiconductor substrate and an optical portintegrated with the semiconductor substrate. The optical port receives aRaman pump beam from an optical fiber. The PIC also includes a firstfilter, integrated with the semiconductor substrate and coupled to theoptical port, to transmit the Raman pump beam and to reject fluorescenceinduced in the optical fiber by the Raman pump beam. The PIC furtherincludes a sample waveguide, integrated with the semiconductor substrateand coupled to the first filter, to receive the Raman pump beam, toexcite a sample in optical communication with the sample waveguide withat least a portion of the Raman pump beam via evanescent coupling, andto receive a scattering signal from the sample in response to theportion of the Raman pump beam.

In some aspects, a PIC for Raman spectroscopy includes a semiconductorsubstrate and an optical port integrated with the semiconductorsubstrate. The optical port receives a Raman pump beam and fluorescenceinduced by the Raman pump beam from an optical fiber. The PIC alsoincludes a directional coupler, integrated with the semiconductorsubstrate and having a first port, a second port, and a third port. Thedirectional coupler receives the Raman pump beam at the first port andoutputs the Raman pump beam at the second port. The PIC further includesa sample waveguide integrated with the semiconductor substrate andcoupled to the second port. The sample waveguide guides the Raman pumpbeam and the fluorescence in a first direction, and excites a sample inoptical communication with the sample waveguide with the Raman pump beamvia evanescent coupling. The sample waveguide also receives a scatteringsignal from the sample in response to the excitation, and guides thescattering signal in a second direction opposite from the firstdirection, and can then couple the scattering signal to a detector.

Some aspects are also directed to a bioreactor having a PIC disposedtherein. Some aspects are also directed to a Raman spectroscopy systemthat includes a PIC as disclosed herein, and further includes an opticalunit removably coupled to the PIC. The optical unit includes a lightsource coupled to the optical port via the optical fiber to launch theRaman pump beam into the optical fiber. The optical unit furtherincludes a detector coupled to the second filter and to receive anddetect the scattering signal transmitted by the second filter.

In some aspects, a method for Raman spectroscopy includes receiving aRaman pump beam via an optical fiber, which also includes receivingfluorescence induced in the optical fiber by the Raman pump beam. Themethod further includes transmitting, via a first filter, the Raman pumpbeam, including rejecting, by the first filter, the fluorescence inducedin the optical fiber by the Raman pump beam. The method also includesreceiving the Raman pump beam in a sample waveguide and exciting asample in optical communication with the sample waveguide with at leasta portion of the Raman pump beam via evanescent coupling. The methodalso includes receiving, via the sample waveguide, a scattering signalfrom the sample in response to the portion of the Raman pump beam, andtransmitting, via a second filter, the scattering signal while blockingtransmission of the remaining portion of the excitation beam.

In some aspects, a method for Raman Spectroscopy includes receiving,from an optical fiber, a Raman pump beam and fluorescence induced by theRaman pump beam in the optical fiber. The method also includes guiding,via a sample waveguide, the Raman pump beam and the fluorescence in afirst direction, and exciting a sample in optical communication with thesample waveguide with the Raman pump beam via evanescent coupling. Themethod further includes receiving, via the sample waveguide, ascattering signal from the sample in response to the excitation, andguiding, via the sample waveguide, the scattering signal in a seconddirection opposite from the first direction.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS

The skilled artisan will understand that the drawings primarily are forillustrative purposes and are not intended to limit the scope of theinventive subject matter described herein. The drawings are notnecessarily to scale; in some instances, various aspects of theinventive subject matter disclosed herein may be shown exaggerated orenlarged in the drawings to facilitate an understanding of differentfeatures. In the drawings, like reference characters generally refer tolike features (e.g., functionally similar and/or structurally similarelements).

FIG. 1 is an example energy diagram depicting changes in frequency ofRaman scattered light that correspond to different molecular energylevels.

FIG. 2 illustrates an example system including an optical unit andmultiple photonic integrated chips (PICs). Specifically, the systemconstitutes an example sensing platform with an optical control unithousing the fiber-integrated optical components andinexpensive/replaceable silicon nitride photonic chips that can beplaced in multiple single-use bioreactors or in series forcontinuous-flow measurements. Each photonic chip can be in a differentbioreactor. In some cases, two or more photonic chips can be in the samebioreactor, and the sensing regions of each respective photonic chip canbe functionalized in the same way, or differently. Optical multiplexersand de-multiplexers can include commercially available MEMS-basedoptical switches.

FIG. 3A is an example photonic integrated chip (PIC) with an on-chipRaman sensing probe to efficiently collects backwards scattered Ramanlight, eliminating the need for on-chip filtering of the laser signaland suppressing the background fluorescence and Raman light generated inthe input/output optical fibers. The Raman sensing probe includes a 2×1component, such as a directional coupler, for controlling light flow.

FIG. 3B is an example PIC similar to FIG. 3A but including a Ramansensing probe using a 2×2 component for controlling light flow, as wellas an additional sensing region and an additional output waveguide.

FIG. 4 is a microscope image of different kinds of visible/near-infraredoptical filters and waveguides fabricated on a silicon nitride photonicsplatform.

FIG. 5 is a scanning electron micrograph images of a Si₃N₄ waveguidedistributed Bragg reflector fabricated at MIT for filtering out thelaser wavelength. The inset plot illustrates the transmissioncharacteristics of the waveguide that enable its operation as a notchfilter.

FIG. 6A is a perspective view of an example flow-integrated Ramanspectroscopic sensor/chip showing details of analyte flow and opticalcoupling. The rigid casing of the sensor and the tight seal created byan O-ring can enable spectroscopic sensing of pharmaceuticals at hightemperatures and pressures.

FIG. 6B is another perspective view of the sensor of FIG. 6A.

FIG. 6C illustrates multiple sensors/chips similar to that in FIG. 6Aarranged in tandem, with the sample outflow of a first chip being thesample inflow into the next chip, and so on.

FIG. 7A illustrates an example Raman chip integrated with a small formatSartorius 250 mL ambr disposable bioreactor.

FIG. 7B illustrates a Sartorius bioreactor workbench that can house upto 24 reactor setups including a reactor integrated with a Raman chip asillustrated in FIG. 7A.

DETAILED DESCRIPTION

All combinations of the foregoing concepts and additional conceptsdiscussed in greater detail below (provided such concepts are notmutually inconsistent) are part of the inventive subject matterdisclosed herein. In particular, all combinations of claimed subjectmatter appearing at the end of this disclosure are part of the inventivesubject matter disclosed herein. The terminology used herein that alsomay appear in any disclosure incorporated by reference should beaccorded a meaning most consistent with the particular conceptsdisclosed herein.

Aspects of the Raman spectroscopy chips disclosed herein encompass smallscale, modular, scalable design while reducing fluorescence (due tooptical fibers for light delivery) from optical coupling to externallight sources, and yet achieving higher SNR than conventional,free-space Raman spectrometers. An unreactive, bio/chemi-compatibleexterior permits for use in a wide variety of environments, from harshchemical settings to sensitive biological environments. Further, asimple optical design that embeds or integrated most components in asemiconductor substrate, while employing simple fiber-optic filteroperation or filter-free, wavelength-independent operation (e.g., seeFIGS. 3A-3B, explained in more details later), is easily andcost-effectively reproduced and tweaked for flexible use.

Example System Design Aspects

As disclosed herein, on-chip photonic spectroscopy can be used forchemical sensing in biological and/or chemical environments. On-chipphotonic spectroscopy can be implemented with on-chip sensing andspectral filtering to overcome the fiber fluorescence challengeassociated with WERS. Additionally, on-chip photonic spectroscopy can beused in a system that comprises multiple low-cost sensing probes and isfully compatible with continuous manufacturing and single-usetechnologies.

Aspects disclosed herein can leverage mature photonics capabilities tominiaturize Raman spectroscopy, a gold-standard technique forselectively identifying and quantifying biological and chemical speciesin complex environments. Chip-integrated, real-time Raman sensors asdisclosed herein can enable faster and more efficient production ofpharmaceuticals with continuous manufacturing and single-usetechnologies. In addition to size reduction, this can provide severalbenefits, including:

-   -   a. Improved robustness (there are no free-space optical        components requiring re-alignment due to integration with a        semiconductor substrate).    -   b. Better reliability (high chip-to-chip uniformity).    -   c. Enhanced light-matter interaction at the surface of the chip        due to evanescent coupling means sensitivities >100 times higher        than free-space Raman spectrometers are possible.    -   d. Large cost reduction by utilizing existing high-volume        silicon CMOS tools.    -   e. The sensors' chemical and biological sensing capabilities        leverage multiple technologies in integrated photonics, such as        tunable light sources, tunable filtering, and tunable detection,        to enable biological and chemical monitoring.    -   f. Waveguide-enhanced Raman scattering (WERS) in single-mode        optical waveguides    -   g. On-chip filtering of background noise from fiber        fluorescence.    -   h. Chip-scale photonic packaging capabilities. The packaged        photonic chip can have epoxy-bonded fibers and can be simply        dropped into aqueous cell-cultures or integrated in tubing for        real-time monitoring of chemical constituents (illustrated in        FIGS. 6A-6C, 7A-7B, described in greater detail later).

A single optical control unit/module can be connected via optical fibersto one or many photonic chips/sensing probes (e.g., as illustrated inFIG. 2, described in greater detail later) that are placed indifficult-to-reach production environments. The optical control unit canhouse components such as a laser, spectrometer, free-space filters, andoptical multiplexer/de-multiplexers, while the photonic chip can includeintegrated spectral filters (e.g., for suppressing background fiberfluorescence) and a sensing region where the tightly confined opticalmode interacts with neighboring chemical species. The system can alsofilter out background fluorescence and Raman background light generatedin the optical fibers to and/or from the chip.

In the Raman sensing system disclosed here (e.g., the system 200 in FIG.2), light from a single optical control unit can be delivered tophotonic chips (via optical fiber), where it is spectrally filtered andexposed to chemicals and biological species that are in direct contactwith the chip surface. The resulting Raman scattered light isefficiently coupled back into the waveguide and co-propagates with theexcitation laser light towards a second filter (e.g., a distributedBragg grating notch filter) that rejects the laser signal. The remainingStokes-shifted Raman light then travels via a second optical fiber backto the optical control unit, where it is filtered again and thenanalyzed using a commercial spectrometer with cooled linear CCD array orsimilar detector.

The photonic chips can be made using a reliable silicon nitride photonicfabrication process and a custom process design kit (PDK). An exampleprocess flow can include a single electron-beam lithography write stepto reliably define optical components with dimensions below 50 nm (e.g.,including, but not limited to, filters, directional couplers, edgecouplers, slot waveguides, strip-to-slot converters, and/or the like)and post-processing to both expose waveguide regions tochemical/biological species, and to define etched waveguide facets forrobust edge-coupling and fiber integration. Delivering light to/from thephotonic chip via optical fiber (in-between the free-space notch andlaser-line filters) produces significant background fluorescence andbackground Raman from the silica fiber. On-chip filters to suppress thisbackground fluorescence and background Raman (e.g., see FIGS. 4-5,described in greater detail later) and further boost the detectionsensitivity.

A single optical control unit can be connected in parallel to manylow-cost sensor chips (e.g., see FIG. 2). The optical control unit caninclude an optical multiplexer/de-multiplexer (e.g., the multiplexer246, the demultiplexer 238) that directs laser light to each low-costsensor chip one-at-a-time. Despite this happening serially, the time toacquire a single spectrum on each probe can be on the order of seconds,which is orders of magnitude faster than the acquisition and samplepreparation times of conventional analytical techniques. Forcell-culture applications, such as on a Sartorius ambr250 24-bioreactorworkstation as shown in FIG. 7B, each disposable bioreactor can includea sterile, miniature probe 710 attached to the inner wall beforecell-culturing. In continuous-flow manufacturing applications, it can bevaluable to obtain information about the chemical concentrations alongthe distance of the tubing and so connecting many of the Raman-sensorsin-line with the flow-tubing may provide real-time data duringproduction (see FIG. 6C). Furthermore, tethering many inexpensive probesto a single unit can dramatically reduce the overall price per sensor.

With such a Raman spectroscopy system, the data acquisition process canbe automated. The acquired data can be fed back into existing controlsfor pharmaceutical production. This means that one can find out duringwhich minute of the day an abnormality occurred in processing, or atwhat exact time cells began producing a compound of interest and takeappropriate actions immediately. Distributed spectroscopic sensornetworks can paint a more complete picture of the pharmaceuticalproduction process, providing information for more efficiently and morereliably developing new drugs.

Example Raman Spectroscopy Systems

FIG. 2 illustrates an example Raman spectroscopy system 200 thatincludes a set of photonic integrated chips (PICs) 210 a-210 n, usefulfor performing Raman spectroscopy as described herein. Explained hereonwith respect to the PIC 210 a for simplicity, each such PIC can includea semiconductor substrate 202 on which one or more components can beembedded. The substrate 202 can be formed of any suitable materials suchas, for example, silicon nitride, titanium dioxide, silicon carbide,silicon, indium phosphide, combinations thereof, and/or the like. Thesubstrate 202 at least in part can be formed of a material that isoptically transparent at the Raman pump beam wavelength(s) and at theRaman scattering wavelength(s) to reduce propagation loss.

The PIC 210 a can also include an optical port 204 integrally formedwith or on the substrate 202 for coupling to an optical fiber 240 a,which can deliver a Raman pump beam from an excitation source (e.g., thelaser 232, described below) to the PIC 210 a. The port 204 can includeany suitable connector for coupling the fiber 240 a such as, forexample, screw-type, clip type, snap type, push-pull type, and/or thelike.

Coupled to the port 204 is a first filter 212 (illustrated here as a“laser line filter”) that is integrated with the substrate 202. Thisfilter 212 can also be integrated into or coupled the end of the fiber240 a coupled to the port 204. The filter 212 can transmit the Ramanpump beam received from the fiber 240 a and reject fluorescence inducedin the fiber 240 a by the Raman pump beam. In other words, the filter212 can transmit light at the wavelength of the Raman pump beam andattenuate or reflect light at other wavelengths, excluding thefluorescence as the Raman pump beam and the fluorescence traverse thefilter 212. The fluorescence can arise, for example, due to the Ramanpump beam interacting with the silica materials of the fibers and canswamp or hide the Raman signal from the sample. As an example, thecenter wavelength of the filter 212 can be about the same as thewavelength of the Raman pump beam. The suppression of other wavelengthscan be high as possible/permissible, such as, for example, up to 60 dB,or greater than 60 dB. The width of the passband of the filter can bewide enough to accommodate for shifts or variation in the laserwavelength, while narrow enough to cut off fluorescence and/or otherspontaneous emission. In some cases, the width of the passband can be upto 5 nm, or greater than 5 nm.

The PIC 210 a can include a sample waveguide (not shown, illustrated anddescribed in greater detail with respect to FIGS. 3A-3B) underlying thesensing region 216, such as, for example, a single-mode optical fiber.The sample waveguide can be coupled to the filter 212 and also beintegrated with the substrate 202. The waveguide receives the filteredRaman pump beam from the filter 212 and, by virtue of its proximity tothe sensing region 216, excites a sample disposed on the region 216 andin optical communication with the sample waveguide with at least aportion of the Raman pump beam via evanescent coupling. The sample inthe sensing region 216, or the vicinity thereof, can generate ascattering signal (e.g., due to Raman scattering) in response to theRaman pump beam, at least a portion of which is received by thewaveguide. This scattering is isotropic; that is, unless occluded, thescattering signal propagates in all directions from the sample. At leasta portion of the scattering signal evanescently couples into thewaveguide and propagates along the waveguide in the same direction asthe remainder/rest/remainder portion of the Raman pump beam and/or inthe opposite direction as the Raman pump beam.

The PIC 210 a also includes a second filter 214 (illustrated here as a“notch filter”) that is integrated with the substrate 202. The filter214 can also be integrated into or coupled to an optical fiber coupledto the PIC 210 a. The second filter 214 is coupled to the samplewaveguide and received the remainder portion of the Raman pump beam aswell as the portion of the scattering signal. The second filter 214 canoperate as a notch filter which blocks light at the wavelength(s) of theRaman pump beam and transmits light at other wavelengths, including theRaman signal wavelength(s). In this manner, the filter 214 can transmitthe scattering signal to a detector, such as the spectrometer 242 viathe fiber 240 b for spectroscopic detection. As an example, the notchband of the filter 214 can be about the same as the wavelength of theRaman pump beam to achieve suppress as high as possible/permissible,such as, for example, up to 60 dB, or greater than 60 dB. The width ofthe notch band of the filter 214 can be wide enough to fully orsubstantially reject the laser, while narrow enough to prevent more thanminimal or acceptable loss of the (useful) scattering signal. In somecases, the width of the notch band can be up to 5 nm, or greater than 5nm.

As also illustrated in FIG. 1, the system 200 also includes an opticalunit/module 230 that can be removably coupled to the PICs 210 a-21Onsuch as by, for example, engaging/disengaging the connectors on the PICsfor the fibers 240 a, 240 b. The optical unit 230 can also encompass anintegrated chip-type architecture. The optical unit 230 includes a lightsource 232 (shown here as a laser) that couples to the optical port 204via the optical fiber 240 a and generates the Raman pump beam. The lightsource 232 can be coupled to an optical isolator 234 that permitsone-way transmission of the Raman pump beam (the source 232 and theisolator 234 can be modular fiber-coupled components or combined in asingle fiber-coupled component). The light source 232 launches the Ramanpump beam into the optical fiber 240 a. The optical unit 230 can also(optionally) include a filter 236 that can be similar to the filter 212,and function to remove any amplified spontaneous emission and/or otherartifacts arising during the Raman pump beam generation on the opticalunit itself.

On the detection side, the optical unit 230 can include a detector 242(shown here as a spectrometer) for detecting the Raman scatteringsignal. Also illustrated is a (optional) filter 244 that can be similarto the filter 214 and transmits light at wavelengths other than that ofthe Raman pump beam.

When the optical unit 230 is to be employed with multiple PICs, it canalso incorporate a de-multiplexer 238 between the source 232 and thePICs 210 a-210 n, and a multiplexer 246 between the scattering signaloutputs of the PICs 210 a-210 n and the detector 242. The de-multiplexer238 and the multiplexer 246 can include, for example, MEMS-based opticalswitches for temporal multiplexing and de-multiplexing. They can also beimplemented as wavelength-based devices for sending Raman pump light atdifferent wavelengths to different PICs 210 a-21On. (Raman pump light atdifferent wavelengths can be generated by tunable light source, such asa tunable laser; a broadband light source, such as a superluminescentdiode; or multiple light sources, such as lasers lasing at differentwavelengths.)

FIG. 3A illustrates a PIC 310 useful for Raman spectroscopy. Unlessnoted otherwise, similarly named components may be structurally and/orfunctionally similar to those describes for the PIC 202 a. The PIC 310can interface with external optical components, such as those describedfor the optical unit 230, for receiving a Raman pump beam and forspectroscopic detection.

The PIC 310 includes a semiconductor substrate 302 that includes anoptical port 304 integrated with the substrate 302. The optical port 304receives a Raman pump beam and fluorescence induced by the Raman pumpbeam from an optical fiber (not shown) and can operate in a mannersimilar to the optical port 204.

The PIC 310 also includes a wavelength selective, multi-port opticalcomponent 314, shown here as a directional coupler 314 with a first port316 a, a second port 316 b, and a third port 316 c. The coupler 314 canhave, as a non-limiting example, a bandwidth greater than about 100 nmand exhibit an extinction ratio up to about 65 dB, with an insertionloss of <1 dB (in addition to any loss (e.g., about 3 dB) incurred bycollecting at one output port, such as the third port 316 c). Thecoupler 314 can be integrated with the substrate 302 and is wavelengthselective in the sense that it has different splitting ratios at theRaman pump and Raman signal wavelengths. The Raman pump beam and thefluorescence can be coupled via the port 314 into the first port 316 a.By virtue of the wavelength selectiveness of the coupler 314, at leasthalf of the Raman pump beam (e.g., 50%, 90%, or 99%) is output at thesecond port 316 b, where a sample waveguide 318 is coupled to the secondport 316 b. The fluorescence from the fiber is also coupled to thesample waveguide 318 via the second port 316 b.

The sample waveguide 318 is illustrated here in spiral form, which canincrease the interaction between the waveguide and a sample whilereducing or minimizing the surface area of the substrate that isassociated with, and given over to, the waveguide 318. The waveguide 318guides the Raman pump beam and the fluorescence in a first directionalong the waveguide, i.e., away from the second port 316 b. The Ramanpump beam can excite a sample in optical communication (e.g., overlayingor flowing past) with the waveguide 318 via evanescent coupling. Theresulting scattering signal can be received by the waveguide 318, andsome portion thereof is coupled into the waveguide 318 and propagates ina second direction along the waveguide 318 that is opposite to the firstdirection, i.e., towards the second port 316 b. (The waveguide 318 mayguide some Raman-scattered light in the first direction too.) In otherwords, at least some of the collected Raman signal counter-propagatesalong the waveguide 318 toward the directional coupler 314.

The scattering signal that propagates in the second direction can thenbe received (at the second port 316 b) and then output (by the thirdport 316 c) by the coupler 314 to an output waveguide 320. Depending onthe directional coupler's wavelength selectivity, 50%, 90%, 99% or moreof the scattering signal is coupled out of the third port 316 c, withthe remainder coupled out of the first port 316 a. In this manner, anyremaining portion of the Raman pump beam and the fluorescence thatpropagates in the sample waveguide in the first direction is neverreturned to the second port 316 b and is effectively filtered outwithout the use of any filter components like those in the PIC 210 a.The output waveguide 320 similarly does not include any filtercomponents, but nevertheless provides at its output the desiredscattering signal, which in turn can be provided to a detector (e.g.,the detector 242) via appropriate coupling means. The effectiverejection of the Raman pump beam and the fluorescence can be on theorder of up to 65 dB, or higher than 65 dB.

FIG. 3B illustrates a PIC 310′ that uses more of the Raman pump lightthan the PIC 310 thanks to a fourth port 316 d′ on the directionalcoupler 314′ that is coupled to an additional/second sample waveguide318 b (the first sample waveguide is reference no. 318 a here). Thecoupling ratios for the coupler 314′ can be changed by changing one ormore of the gap, width, or length of the coupling region, of thewaveguide, or both. In this setup, the first sample waveguide 318 areceives (i.e., the directional coupler 314′ couples) a first portion(e.g., 50%) of the Raman pump beam and a first portion of thefluorescence, while the second sample waveguide 318 b receives theremainder (e.g., 50%) of the Raman pump beam and a second/remainderportion of the fluorescence. This ensures that all of the Raman pumpbeam excites the sample (neglecting insertion loss, attenuation, etc.).During operation, both waveguides 318 a, 318 b excite the sample andreceive counter-propagating scattering signals. The directional coupler314′ couples the (first) scattering signal from the first waveguide 318a and the (second) scattering signal from the second waveguide 318 bthrough the output waveguide 320′ to output 322′, which may be coupledto a detector (not shown). Again, because the scattering signalspropagate in the opposite direction as the Raman pump beam andfluorescence from the input fiber, they are not present at the output322′. The effective rejection of the Raman pump beam and thefluorescence can be on the order of up to 65 dB, or higher than 65 dB.In this manner, signal collection from the sample can be increased bythe PIC 310′ relative to the PIC 310 which can lose half the Raman pumpbeam at the directional coupler 314. In the PIC 310′, on the other hand,the entire Raman pump beam can be consumed and used to excite thesample.

As noted above for the PIC 210 a, one or more PICs 310 and/or 310′ canbe removably coupled to an optical unit including excitation anddetection components, such as the optical unit 210. As an example, theinput ports 304, 304′ can be coupled to the optical unit 210 via thedemultiplexer 238, and the outputs 322, 322′ can be coupled to theoptical unit via the multiplexer 246.

FIG. 4 illustrates example fabrication of different components of thePICs 210 a, 310, 310′ on a silicon nitride photonics platform/substrate.Waveguides 410 are wrapped in a bow-tie manner and can provide analternative design to the spiral form of the waveguides 318, 318 a, 318b. The notch filters 412 are in a substantially linear configuration,and the filters 214, 244 can be designed and/or laid out in this manner.The laser-line filters 414 are in an elongated looped configuration, andthe filters 212, 236 can be designed and/or laid out in this manner. Theresonant filters 416 form a series of circular loops and can be used todesign the filters 212, 214 described herein.

FIG. 5 shows detail of an example, silicon waveguide distributed Braggreflector for filtering out the laser wavelength (e.g., similar to theoperation of the filters 214, 244) via reflection while permitting theRaman scattering signal to propagate. This example design is a comb-likeconfiguration.

FIGS. 6A and 6B illustrate a Raman sensing system 600 with a replaceable(and optionally disposable) PIC 610 (e.g., similar to the PICs 210 a,310, 310′) useful in fluid tight settings with controlled sample flow.The sensing system 600 includes and/or is couplable to a housing 604that provides a fluid tight compartment for the various componentswithin. As illustrated in FIGS. 6A-6B, an opening 614 can provide foroptical coupling of the PIC 610 to external components (e.g., forcoupling the fibers 240 a, 240 b). A sample holding region 606 withinthe housing is in optical communication with the PIC 610 and holds thesample during analysis. A sample input port 616 is formed on the surfaceof the housing 604 and is coupled to the sample holding region 606 topermit inflow of the sample for analysis with the PIC 610. At the otherend of the sample holding region 606 (best illustrated in FIG. 6B) asample output port 612 is formed on the surface of the housing 604 andis coupled to the sample holding region 606 to permitoutflow/circulation of sample.

FIG. 6C illustrates multiple Raman sensing systems 600 a-600 n, eachwith a different PIC (not shown), that can be used for multi-pointmeasurements on the same sample. As illustrated in FIG. 6C, the samplecan be input into the Raman sensing system 600 a via its sample inputport, and then output via its sample output port and into the sampleinput port of the Raman sensing system 600 b, and so on. Multiple,redundant measurements can be made possible when each of the Ramansensing systems 600 a-600 n and PICs is configured the same way (e.g.,has the same filter characteristics). Additionally, differentmeasurements on the same sample can be made possible when each of theRaman sensing systems 600 a-600 n is configured differently (e.g.,employs different excitation wavelengths).

FIG. 7A illustrates an example PIC design 710 (e.g., each similar to thePICs 210 a, 310, 310′) useful for sample immersion measurements. In suchsetups, the PIC 710 is immersed in the sample, with the sample waveguidebordering or interfacing with an external surface of the PIC'ssemiconductor substrate (e.g., the substrate 202, 302) exposed to thesample. The exposed, external surface can include a coating to preventadhesion of the sample, to maintain a sterile environment by preventingmicrobial growth, and/or the like. The coating can include a fluorinatedpolymer, aluminum oxide, and/or the like.

Accordingly, aspects disclosed herein can be directed to bioreactordevices, such as the bioreactor 720 illustrated in FIG. 7A, that have aPIC 710 (e.g., similar to the PICs 210 a, 310, 310′) disposed thereinsuch as for, for example, for monitoring the microenvironment of thebioreactor. FIG. 7B illustrates a series of bioreactors similar to the720 each including the photonic chip 710 disposed therein.

Aspects disclosed herein can also be directed to continuous flowsystems, such as flow cytometry for example, where a PIC (e.g., similarto the PICs 210 a, 310, 310′) can be positioned inline with flow forcontinuous sample analysis.

Example Raman Spectroscopy Methods

Aspects disclosed herein can also be directed to methods for Ramanspectroscopy using, for example, a PIC similar to the PIC 210 a. Themethod can include receiving a Raman pump beam (e.g., generated by thelight source 232) via an optical fiber (e.g., the fiber 240 a), whichalso includes receiving fluorescence induced in the optical fiber by theRaman pump beam. The method further includes transmitting, via a firstfilter (e.g., the filter 212), the Raman pump beam, and rejecting, bythe first filter, the fluorescence induced in the optical fiber by theRaman pump beam. The method also includes receiving the Raman pump beamin a sample waveguide (e.g., the waveguide 318, 318 a, 318 b) andexciting a sample in optical communication with the sample waveguidewith at least a portion of the Raman pump beam via evanescent coupling.The method also includes receiving, via the sample waveguide, ascattering signal from the sample in response to the portion of theRaman pump beam, and transmitting (e.g., to the detector 242), via asecond filter (e.g., the filter 214), the scattering signal whileblocking transmission of the remaining portion of the excitation beam.

The method can also encompass pumping a sample into (e.g., via thesample input port 610) a sample holding region (e.g., the sample holdingregion 606) in optical communication with the sample waveguide prior toexciting the sample. The method can also encompass pumping out (e.g.,via the sample output port 612) the sample after exciting the sample.

Aspects disclosed herein can also be directed to methods for Ramanspectroscopy using, for example, a PIC similar to the PIC 310, 310′. Themethod includes receiving, from an optical fiber (e.g., at the port 304,via the fiber 240 a), a Raman pump beam and fluorescence induced by theRaman pump beam in the optical fiber. The method also includes guiding,via a sample waveguide (e.g., the waveguide 318, 318 a, 318 b), theRaman pump beam and the fluorescence in a first direction and exciting asample in optical communication with the sample waveguide with the Ramanpump beam via evanescent coupling. The method further includesreceiving, via the sample waveguide, a scattering signal from the samplein response to the excitation, and guiding, via the sample waveguide,the scattering signal in a second direction (e.g., towards the secondport 316 b, 316 b′) opposite from the first direction, and towards adetector (e.g., the detector 242).

The method can also encompass guiding, via a second sample waveguide(e.g., the waveguide 318 b, where the waveguide 318 a is a firstwaveguide), a second portion of the Raman pump beam and a second portionof the fluorescence in the first direction (i.e., away from the fourthport 318 d). The method can also include exciting the sample in opticalcommunication with the second sample waveguide with the second portionof the Raman pump beam via evanescent coupling, and receiving a secondscattering signal from the sample in response. The second scatteringsignal is guided in the second direction (i.e., towards the fourth port318 d), and then guided towards the detector.

Aspects disclosed herein can also be directed to methods for Ramanspectroscopy using, for example, a PIC similar to the PIC 210 a, 310,310′. The method can include guiding a Raman pump beam with an opticalfiber (e.g., the fiber 212), the Raman pump beam inducing fluorescencein the optical fiber. The method can further include coupling the Ramanpump beam and the fluorescence to a photonic integrated circuit (PIC,similar to the PIC 210 a, 310, 310′) via the optical fiber. The methodcan also include exciting a sample in optical communication with the PICwith the Raman pump beam via evanescent coupling, such that the Ramanpump beam inducing Raman scattering in the sample. The Raman scatteringis collected via the PIC, and the Raman pump beam and the fluorescenceare filtered, also via the PIC, from the Raman scattering. In somecases, the coupling can include coupling the Raman pump beam and thefluorescence to a filter (e.g., the filter 212) of the PIC (e.g., thePIC 210 a) that transmits the Raman pump beam and rejects thefluorescence. In some cases, the filtering can include guiding the Ramanscattering propagating along a second direction (e.g., towards thesecond port 318 b and/or the fourth port 318 d) opposite to a firstdirection of propagation of the Raman pump beam and the fluorescence.

Example Features

An example on-chip photonic Raman spectroscopy system can detectchemical and biological species while simultaneously suppressing thefluorescent light and Raman scattered light produced from the opticalfibers. The example system may include one or more of the followingfeatures:

-   -   (1) A chip-scale Raman sensing probe with the following discrete        elements (e.g., see FIG. 2):        -   a. A fiber-to-chip coupling region, wherein light from the            optical fiber (e.g., the fiber 240 a) is transmitted to a            single-mode photonic waveguide.        -   b. An on-chip filter (e.g., the filter 212) after the            fiber-to-chip coupling region that serves to transmit the            laser wavelength and block light at wavelengths above and            below the laser wavelength.        -   c. A waveguide sensing region (e.g., the region 216 having a            sample waveguide) after the first on-chip filter, wherein            evanescent light from the waveguide mode scatters with            chemicals of interest and is collected back by the            waveguide.        -   d. An on-chip filter (e.g., the filter 214), after the            waveguide sensing region, that transmits light at            wavelengths above or below the laser wavelength and blocks            light in a wavelength band that includes the laser            wavelength.        -   e. A chip-to-fiber coupling region, wherein light from the            single mode photonic waveguide is transmitted to an optical            fiber (e.g., the fiber 240 b).    -   (2) A chip-scale Raman sensing probe with the following discrete        elements (e.g., see FIGS. 3A-3B):        -   a. A fiber-to-chip coupling region, wherein light from the            optical fiber is transmitted to a single-mode photonic            waveguide.        -   b. A 4-port optical component (such as an adiabatic 2×2            directional coupler or a 2×2 multi-mode interferometer, see            the coupler 314′), which couples light from the            fiber-to-chip coupling region to two output waveguides via            the top input (e.g., the port 316 a). Alternatively, this            component can be a 3-port device (such as a 2×1 directional            coupler or 2×1 multi-mode interferometer, see the coupler            314) that couples light from fiber-to-chip coupling region            to the single output waveguide via the top input (e.g., via            the ports 316 a, 316 b).        -   c. One (e.g., a region associated with the waveguide 318) or            two waveguide (e.g., a region associated with the waveguides            318 a, 318 b) sensing regions after the 2×2 or 2×1            component, wherein evanescent light from the waveguide mode            scatters with chemicals of interest that is collected in the            waveguide and propagates in the opposite direction as the            light from directional coupler. The Raman light then travels            back to the 2×2 or 2×1 directional coupler, where some of            the light travels to the bottom input waveguide.        -   d. A waveguide (e.g., the output waveguide 320, 320′) that            connects the bottom input of the 2×2 or 2×1 directional            coupler to a chip-to-fiber optical coupling region, wherein            light from the single mode photonic waveguide is transmitted            to an optical fiber.    -   (3) A Raman spectroscopy system (e.g., see FIGS. 2, 3A-3B) that        includes:        -   a. One or more of the chip-scale Raman sensing probes (e.g.,            the PIC 210 a, 310, 310′) described immediately above.        -   b. One or more single-mode optical fibers (e.g., the fibers            240 a, 240 b) that connect and deliver light to/from the            optical control/unit described immediately below with each            Raman sensing probe.        -   c. An optical control unit/module with the following            elements:            -   i. A single-frequency light source (e.g., the source                232), such as a 780 nm continuous-wave laser (or a laser                at any other wavelength)            -   ii. (optional) An optical isolator (e.g., the isolator                234), after the single-frequency light source (either a                separate module or one integrated with the                single-frequency light source)            -   iii. (optional) A polarization control module (not                shown) that corrects for small polarization differences                between the output laser and the desired optical                polarization of the photonic chip's waveguide            -   iv. (optional) A free-space or fiber-integrated optical                filter (sometimes called a laser-line filter', e.g., the                filter 236), that has high transmission at the laser                wavelength and low transmission at wavelengths above and                below the laser wavelength. This can suppress amplified                spontaneous emission that is a large source of                background noise in Raman spectroscopy systems            -   v. (optional) An optical demultiplexer (e.g., the                demultiplexer 238), comprising one or more optical                switches, that directs light from the one laser source                to one or many optical fibers that are each connected to                the photonic chips.            -   vi. (optional) An optical multiplexer (e.g., the                multiplexer 246) that receives as input one fiber from                each of the photonic chips and directs the light to a                single optical fiber output.            -   vii. (optional) A free-space or fiber-integrated optical                filter (sometimes called a ‘notch-filter’, e.g., see the                filter 244) that has high transmission at wavelengths                above and below the laser wavelength and low                transmission at the laser wavelength.            -   viii. A spectrometer (e.g., the detector 242) that                receives the Raman-scattered optical signal(s) and                measures the intensity at each wavelength of interest                corresponding to the Raman-scattered optical signal.    -   (4) The fiber terminations at the chip-coupling region may be:        -   a. flat-cleaved, allowing for low-loss coupling to            low-index-contrast waveguides due to reduced mode-field            diameter mismatch;        -   b. tapered and lensed;        -   c. in a linear fiber array made of flat-cleaved fibers            mounted in v-grooves;        -   d. tapered and pitch-reduced (e.g., forming a Pitch Reducing            Fiber Optical Array); and/or        -   e. coupled to a mode-converting interposer chip.    -   (5) A chip-scale Raman sensing probe as above with coupling        regions that comprise one or more of:        -   a. inverse tapered couplers at the edge of the chip;        -   b. sub-wavelength grating couplers at the edge of the chip;            and/or        -   c. fiber grating couplers that direct light upwards out of            the chip to a fiber.    -   (6) A Raman sensing probe, as above, with optical filters that        are composed of:        -   a. Ring or disk resonators,        -   b. Periodic Bragg reflectors (DBRs), and/or        -   c. Contra-directional couplers.    -   (7) A chip-scale Raman sensing probe as above fabricated using        silicon nitride, titanium dioxide, silicon carbide, silicon,        indium phosphide, or any other material that is optically        transparent in the wavelength region of interest, as the        waveguiding material.    -   (8) A chip-scale Raman sensing probe with sensing region as        above with a thin layer of material on the top for coating,        anti-stick, and sterilization (autoclave or gamma-ray) purposes.    -   (9) A chip-scale Raman sensing probe with a thin layer of        material on the top of the sensing region as immediately above        where the thin layer of material is:        -   a. A fluorinated polymer like PTFE or Teflon; or        -   b. Aluminum oxide Al₂O₃.    -   (10) A Raman spectroscopy system as above where the sensing        probe(s) is (are) integrated in one (e.g., the bioreactor 720 in        FIG. 7A) or more bioreactors (e.g., the multi-bioreactor setup        in FIG. 7B)    -   (11) A Raman spectroscopy system as above where the sensing        probe(s) is (are) integrated in one or more continuous-flow        environments (e.g., the arrangement of Raman sensing systems 600        a-600 n in FIG. 6C).    -   (12) A Raman spectroscopy system as above, where there is an        additional optical polarization controller (not shown) to        control the polarization state of light before it is launched        into the optical fibers.    -   (13) Fibers as above that are polarization-maintaining        single-mode fibers.

Example Applications

Examples of the systems and PICs disclosed herein (e.g., with respect toFIGS. 2-7) can include small and inexpensive sensing/Raman spectroscopydevices to be integrated in pharmaceutical production environments. Oneapplication can be for small-volume cell-cultures and bioreactors (e.g.,see FIGS. 7A-7B) where the chip-integrated sensors can providereal-time, in-situ monitoring of metabolic processes, feed-materialdepletion, and reaction product formation. Another application can beflow-integrated, in-line monitoring of active pharmaceutical ingredientsin continuous manufacturing environments (e.g., see FIG. 6C).

With respect to pharmaceutical use, generally, the production ofcritical ingredients for pharmaceuticals is traditionally performedusing ‘batch-production’ methods in large, multiple-use stainless steelvessels. The traditional batch-production methods requirelabor-intensive cleaning and sterilization of the steel vessels betweenproduction runs and stop-and-go processes that bottleneck productionspeed. Recently, overall trends towards personalized medicine, morepotent drugs, and rapidly growing markets for therapeutic proteins (suchas antibody-based products) is driving demand for smaller productionvolumes, increased process flexibility, and faster production times.This has ignited interest from the pharmaceutical industry in two keyareas: (1) single-use technologies (SUTs), where tubing, vessels,sensors, etc. are disposable and guaranteed to be sterile prior to use,and (2) continuous-flow manufacturing techniques, where an entirepharmaceutical production line is performed using flow-reactions andwithout any stops.

Single-use technologies, despite being disposable, have also been shownto have a positive environmental impact, as the cleaning andsterilization process of stainless-steel vessels is environmentallyworse than the solid-waste generation from SUTs. Continuous productionof pharmaceutical ingredients would dramatically reduce productionfacility sizes, significantly reduce the amount of time to producepharmaceutical ingredients (in some cases from months to days), increaseprocess flexibility, increase reliability, and reduce overall waste,process contamination, and environmental impacts. For this reason, therehas been a growing interest and trend towards these SUTs forpharmaceutical production, and regulatory bodies such as the US FDA havebeen strongly encouraging pharmaceutical companies to transition towardscontinuous manufacturing techniques. However, moving to thesetechnologies alone is not enough to meet the growing demands facingpharmaceutical production environments. It is envisioned that thepharmaceutical facility of the future should have continuous production,100% single-use equipment, closed processing, and “Ballroom” processing.Closed processing refers to never exposing pharmaceuticals or chemicalsto the environment (to prevent contamination and increase yield).“Ballroom” processing refers to having all equipment in one central roomrather than multiple facilities (with larger net space).

To realize these breakthrough pharmaceutical production techniques,process analytical technologies (PATs) should find ways of becomingintegrated with single-use technologies and continuous-flowenvironments. Real-time monitoring of reaction progress is encouraged bythe US FDA and international organizations as a way of promoting GMP(“Good Manufacturing Practices”) and QbD (“Quality by Design”)processes. Recently, the rapid progress towards SUTs and continuousmanufacturing has generated a large demand for improved sensors beyondwhat is currently available. For disposable bioreactors andcontinuous-flow production environments, companies are looking fortechnologies that can provide real-time information on temperature, pH,dissolved oxygen, cell density, viable cell density nutrients, andmetabolite concentrations. A reason for this demand is that current‘gold-standard’ analytical techniques involve batch-testing and longsample prep and wait times (such as hours per sample for chromatographymethods). Furthermore, existing commercial sensors that can beintegrated with single-use technologies include: (1) electrochemicalsensors, which have high false-positive rates and relatively poorselectivity, and (2) optical sensors, which are currentlysingle-wavelength sensors with poor selectivity or expensive andrelatively large spectroscopic probes. Without better sensing techniquesintegrated into future bioproduction facilities, it may be difficult todetect abnormal events during production, optimize new processes, oravoid stop-test-and-go situations that are currently prevalent in theindustry.

Raman spectroscopy in particular is capable of selectively identifyingactive pharmaceutical ingredients by probing the unique spectral‘fingerprint’ for each molecular structure. Raman spectroscopy is usedfor in-line and real-time monitoring. Miniaturized, low-cost Ramansensors as disclosed herein can be deployed in both R&D and productionenvironments. The current testing process for one such environment cangenerally be as follows:

-   -   a. A robotic arm (or human) uses a clean syringe to take a small        amount of liquid out of each of the 24 bioreactors at a fixed        time interval (maybe once every 4-6 hours) and place it in a        96-well (or smaller equivalent) plate. This is because opening        the top and introducing a syringe can be a contamination risk.    -   b. A person or robot moves the plate over to a separate PAT in        the lab (often a mass-spectrometer or liquid chromatography        tool), and marks or remembers which sample corresponds to which        bioreactor.    -   c. Results are returned in a few hours from a large benchtop        tool that measures each sample one-at-a-time.    -   d. The operator analyzes this data.    -   e. This information is then used to determine which bioreactors        are performing well and which bioreactors need additional feed        material, a pH change, etc.

However, by this time it is typically too late to make any changes tothe current batch and so the information is used to inform the next setof experiments or cell-cultures. This iterative process influencesdevelopment worth upwards of $4 M/year for every table-top sized24-reactor workstation (cell-growth and fermentation is a $30 B/yearmarket). There is hence a need for (1) more information about reactionsoccurring in their small cell-cultures, (2) selective identification ofcommon molecules like glucose, acetate, and ethanol andsmall-concentration byproducts, and (3) techniques for obtaining thisinformation that are low-cost and ideally disposable (for sterility).

Raman spectroscopy is capable of selectively identifying activepharmaceutical ingredients by probing the unique spectral ‘fingerprint’for each molecular structure. It can be used for in-line and real-timemonitoring. Existing Raman spectrometers, however, are unable to meetthe demands of process monitoring for SUTs, since the unit cost isprohibitively high (˜$30-40 k per portable Raman spectrometer) and ahuman operator tests each bioreactor one-at-a-time. Integrating achip-scale Raman sensor such as disclosed herein in each small-volumebioreactor or cell-culture vessel can overcome some of these challenges.Having many small, accurate, and low-cost spectroscopic sensorsproviding real-time data in parallel would improve automation of thegrowth process, increase efficiencies (less wasted resources from failedbatches), and reduce time-to-development for new pharmaceuticals anddrugs.

Likewise, continuous-flow manufacturing of pharmaceuticals is a newtechnique for quickly and efficiently producing pharmaceuticals thatrequires new tools and techniques for analysis. In batch-production,samples could be taken to a lab and tested on several highly accurate(albeit high-cost and often slow) instruments. To prevent stops likethis in a continuous manufacturing environment, real-time measurementtools can be integrated with flow-tubing and provide sufficient chemicalinformation. In addition, each point in the process that requiresmonitoring needs its own dedicated measurement tool (rather than onetool in a stop-test-and-go batch environment). To address these needs,future sensors should be low-cost (for monitoring at many points in theprocess), operated in parallel (to simplify analysis and feedback givenlarge amounts of data), and capable of reliable detection.

Currently, spectrophotometric process analytical tools in thepharmaceutical industry are a $185 M/year market. The systems, PICs, andmethods disclosed herein can augment existing spectroscopic analyticaltools and can be used in areas where PATs were previously unable to beapplied.

Conclusion

While various inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

Also, various inventive concepts may be embodied as one or more methods,of which an example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A photonic integrated circuit (PIC) for Raman spectroscopy, the PICcomprising: a semiconductor substrate; an optical port, integrated withthe semiconductor substrate, to receive a Raman pump beam from anoptical fiber; a first filter, integrated with the semiconductorsubstrate and coupled to the optical port, to transmit the Raman pumpbeam and to reject fluorescence induced in the optical fiber by theRaman pump beam; a sample waveguide, integrated with the semiconductorsubstrate and coupled to the first filter, to receive the Raman pumpbeam, to excite a sample in optical communication with the samplewaveguide with at least a portion of the Raman pump beam via evanescentcoupling, and to receive a scattering signal from the sample in responseto the portion of the Raman pump beam; and a second filter, integratedwith the semiconductor substrate and coupled to the sample waveguide, totransmit the scattering signal and to block the remaining portion of theexcitation beam.
 2. The PIC of claim 1, wherein the sample waveguide isin optical communication with the sample via an external surface of thesemiconductor substrate.
 3. The PIC of claim 2, wherein at least aportion of the external surface that is in optical communication withthe sample waveguide includes a coating comprising at least one of afluorinated polymer or aluminum oxide.
 4. The PIC of claim 1, furthercomprising: a housing to provide a fluid-tight compartment within; asample holding region within the housing in optical communication withthe sample waveguide to hold the sample; a sample input port formed onthe surface of the housing and coupled to the sample holding region topermit inflow of the sample into the sample holding region; and a sampleoutput port formed on the surface of the housing and coupled to thesample holding region to permit outflow of sample from the sampleholding region.
 5. The PIC of claim 4, wherein the PIC is a first PIC,and wherein the sample output port of the first PIC is couplable to asample input port of a second PIC.
 6. A bioreactor having the PIC ofclaim 1 disposed therein.
 7. A continuous flow device having the PIC ofclaim 1 disposed inline with a continuous sample flow.
 8. A Ramanspectroscopy system, comprising: the PIC of claim 1; and an optical unitremovably coupled to the PIC, the optical unit including: a lightsource, coupled to the optical port via the optical fiber, to launch theRaman pump beam into the optical fiber; a detector, coupled to thesecond filter, to receive and detect the scattering signal transmittedby the second filter.
 9. The system of claim 8, wherein the PIC is afirst PIC of a set of PICs coupled to the optical unit, the optical unitfurther including: an optical demultiplexer circuit to couple the lightsource to the optical port of each PIC; and an optical multiplexercircuit to couple the detector to the second filter of each PIC.
 10. Aphotonic integrated circuit (PIC) for Raman spectroscopy, the PICcomprising: a semiconductor substrate; an optical port, integrated withthe semiconductor substrate, to receive a Raman pump beam andfluorescence induced by the Raman pump beam from an optical fiber; adirectional coupler, integrated with the semiconductor substrate andhaving a first port, a second port, and a third port, to receive theRaman pump beam at the first port and to output the Raman pump beam atthe second port; a sample waveguide, integrated with the semiconductorsubstrate and coupled to the second port, to guide the Raman pump beamand the fluorescence in a first direction, to excite a sample in opticalcommunication with the sample waveguide with the Raman pump beam viaevanescent coupling, to receive a scattering signal from the sample inresponse to the excitation, and to guide the scattering signal in asecond direction opposite from the first direction; and an outputwaveguide, integrated with the semiconductor substrate and coupled tothe third port of the directional coupler, to guide the scatteringsignal to a detector.
 11. The PIC of claim 10, wherein the directionalcoupler includes a fourth port, wherein the sample waveguide is a firstsample waveguide that receives a first portion of the Raman pump beamand a first portion of the fluorescence, wherein the scattering signalis a first scattering signal, the PIC further including a second samplewaveguide, integrated with the semiconductor substrate and coupled tothe fourth port, to guide a second portion of the Raman pump beam and asecond portion of the fluorescence in the first direction, to excite thesample in optical communication with the second sample waveguide withthe Raman pump beam via evanescent coupling, to receive a secondscattering signal from the sample in response to the excitation with thesecond portion of the Raman pump beam, and to guide the secondscattering signal in the second direction, wherein output waveguideguides the second scattering signal to the detector.
 12. The PIC ofclaim 10, wherein the sample waveguide is in optical communication withthe sample via an external surface of the semiconductor substrate. 13.The PIC of claim 10, further comprising: a housing to provide afluid-tight compartment within; a sample holding region within thehousing in optical communication with the sample waveguide to hold thesample; a sample input port formed on the surface of the housing andcoupled to the sample holding region to permit inflow of the sample intothe sample holding region; and a sample output port formed on thesurface of the housing and coupled to the sample holding region topermit outflow of sample from the sample holding region.
 14. The PIC ofclaim 13, wherein the PIC is a first PIC, and wherein the sample outputport of the first PIC is couplable to a sample input port of a secondPIC.
 15. A system, comprising: the PIC of claim 10; an optical unitremovably coupled to the PIC, the optical unit including: a light sourcecoupled to the optical port via the optical fiber, to launch the Ramanpump beam into the optical fiber; a detector coupled to the outputwaveguide, to receive and detect the scattering signal transmitted bythe output waveguide.
 16. The system of claim 15, wherein the PIC is afirst PIC of a set of PICs coupled to the optical unit, the optical unitfurther including: an optical demultiplexer circuit to couple the lightsource to the optical port of each PIC; and an optical multiplexercircuit to couple the detector to the output waveguide of each PIC. 17.A bioreactor having the PIC of claim 10 disposed therein.
 18. Acontinuous flow system having the PIC of claim 10 disposed inline with asample flow.
 19. A method of Raman spectroscopy, comprising: receiving aRaman pump beam via an optical fiber, including receiving fluorescenceinduced in the optical fiber by the Raman pump beam; transmitting, via afirst filter, the Raman pump beam, including rejecting, by the firstfilter, the fluorescence induced in the optical fiber by the Raman pumpbeam; receiving the Raman pump beam in a sample waveguide; exciting asample in optical communication with the sample waveguide with at leasta portion of the Raman pump beam via evanescent coupling; receiving, viathe sample waveguide, a scattering signal from the sample in response tothe portion of the Raman pump beam; transmitting, via a second filter,the scattering signal while blocking transmission of the remainingportion of the excitation beam.
 20. The method of claim 19, furthercomprising pumping a sample into a sample holding region in opticalcommunication with the sample waveguide prior to said exciting; andpumping the sample out of the sample holding region after said exciting.21. A method of Raman Spectroscopy, comprising: receiving, from anoptical fiber, a Raman pump beam and fluorescence induced by the Ramanpump beam in the optical fiber; guiding, via a sample waveguide, theRaman pump beam and the fluorescence in a first direction; exciting asample in optical communication with the sample waveguide with the Ramanpump beam via evanescent coupling; receiving, via the sample waveguide,a scattering signal from the sample in response to the excitation;guiding, via the sample waveguide, the scattering signal in a seconddirection opposite from the first direction; and guiding the scatteringsignal to a detector.
 22. The method of claim 21, wherein the samplewaveguide is a first sample waveguide that receives a first portion ofthe Raman pump beam and a first portion of the fluorescence, and whereinthe scattering signal is a first scattering signal, the method furthercomprising: guiding, via a second sample waveguide, a second portion ofthe Raman pump beam and a second portion of the fluorescence in thefirst direction; exciting the sample in optical communication with thesecond sample waveguide with the second portion of the Raman pump beamvia evanescent coupling; receiving, via the second sample waveguide, asecond scattering signal from the sample in response; guiding, via thesecond sample waveguide, the second scattering signal in the seconddirection; and guiding the second scattering signal to the detector. 23.A method of Raman spectroscopy, the method comprising: guiding a Ramanpump beam with an optical fiber, the Raman pump beam inducingfluorescence in the optical fiber; coupling the Raman pump beam and thefluorescence to a photonic integrated circuit (PIC) via the opticalfiber; exciting a sample in optical communication with the PIC with theRaman pump beam via evanescent coupling, the Raman pump beam inducingRaman scattering in the sample; collecting the Raman scattering with thePIC; and filtering, via the PIC, the Raman pump beam and thefluorescence from the Raman scattering.
 24. The method of claim 23, thecoupling including coupling the Raman pump beam and the fluorescence toa filter of the PIC that transmits the Raman pump beam and rejects thefluorescence.
 25. The method of claim 23, the filtering includingguiding the Raman scattering propagating along a second directionopposite to a first direction of propagation of the Raman pump beam andthe fluorescence.